The invention concerns providing automated, high throughput protein crystallography.
X-ray crystallography is typically performed by diffracting x-rays through a crystalline sample and determining the resultant pattern of diffracted radiation on a detector or target. Commercially available systems typically involve the use of a goniometer to which the sample is mounted. Goniometers provide accurate angular measurement and control needed for x-ray crystallography, but are also expensive. Further, commercially available goniometers do not provide for rapid and efficient robotic mounting or dismounting of samples. Although mechanisms to mechanically transfer samples to and from goniometer mounts are possible, such mechanisms are cumbersome. Moreover, combining such mechanisms with goniometers in an effort to achieve automation results in an inherently more complex, and therefore less reliable, mechanical system.
One alternative to a goniometer-based x-ray crystallography system is to use independently mounted x-ray source, sample holder, and detector, as described in U.S. Pat. No. 6,064,717 to Ortega, et al. Such a system allows for computer control of the source, holder, and detector positioning, and avoids the necessity of a goniometer-style mounting system.
However, new techniques in biochemistry and protein analysis will require an automated system for performing protein crystallography, so that large numbers of samples may be tested efficiently. Doing so effectively will require as simple a mechanical system as possible, to minimize potential breakdowns and reduce the need for human intervention. Thus, it is desirable to provide a robotic crystallography system which incorporates the use of multiply-independent x-ray source, sample, and detector, and which can also automatically select a sample from a group of samples to be tested, move the sample into position, test the sample, and deposit the sample for later use or disposal.
This procedure is complicated by the need in protein crystallography to regulate the sample temperature by keeping the samples at a controlled, usually reduced, temperature. Samples are generally stored in a temperature-regulated environment, such as a dewar containing liquid nitrogen, prior to and after testing. Further, each sample may be equipped with a portable liquid nitrogen bath which will protect the sample""s temperature while it is being moved from the storage location to the testing location. Such a portable bath must be decoupled from the sample while the sample is being tested, and the sample must be maintained in a temperature controlled state during testing. After testing, it is necessary to recouple the portable nitrogen bath to the sample for transportation back to a protected environment, so that the sample will always be maintained in a temperature controlled state in case the sample is needed for further experimentation.
It is an object of this invention to provide a robotic x-ray crystallography system in which multiple samples may be automatically, selectively tested, and in which the sample selector also provides the function of a goniometer during testing.
It is a further object of this invention to provide such a robotic x-ray crystallography system in which a sample to be tested is coupled and subsequently uncoupled from a robotic selector arm only once during each testing cycle.
It is another object of this invention to protect samples from degradation by maintaining them in a temperature controlled state at all phases of the testing cycle.
Samples used in crystallography, such as protein samples, must often be maintained in an artificial environment, for example, they may be maintained at a substantially reduced temperature. This description is directed, as an example, to maintaining temperature controlled samples at liquid nitrogen temperatures or other selected, reduced temperatures, although those of skill in the art will recognize that other means of controlling temperature are possible, and that such known variations in maintaining artificial environments are incorporated in the scope of this description.
For high-throughput x-ray crystallography, a set of samples will be provided and stored in a controlled environment, such as a first storage dewar containing liquid nitrogen. As those of skill in the art will recognize, many variations on such an arrangement are possible, and the samples can be arrayed in a fixed arrangement, on a conveyor system, or in any other such manner of positioning, or moving the samples into position, as shall be convenient to the purpose of placing the samples in a position where they may be selectively coupled to a robotic selector, and which allows the continuous identification of each sample.
Each sample may be connected to a sample holder, which comprises an extension or other point which may be selectively coupled onto by a robotic grasping device. This grasping point provides a fixed spatial relationship to the sample, so that positioning the sample holder by positioning the grasping point will also fix the spatial location of the sample. Additionally, each sample holder may comprise an integral or attachable collar (or similar connector) to allow the selective coupling and uncoupling of a liquid reservoir to the sample holder in such a fashion that the sample will be contained within the liquid reservoir when it is connected to the sample holder.
To perform high-throughput crystallography on the samples, a robotic arm with a coupling device capable of grasping an individual sample is operated under automatic control, such as by a program stored within the robotic device or on a separate, connected computer. The robotic arm is comprised of known components, such as base, support, wrist, elbow, and hand, in such combination as is necessary to provide the degree of articulation necessary to allow the robotic arm to perform its functions. The robotic hand comprises a grapple capable of securely gripping the grasping point of a sample holder. Once a sample is selected and gripped by the robotic arm, the arm is then moved to the necessary position to serve in the capacity of a goniometer, positioning the sample holder so that the sample will be placed in a known spatial relationship to an x-ray source and a detector.
Because the samples must be maintained at an artificial temperature, provision is also made for protecting the sample during the transition from the initial storage area to the testing area. In the example of using liquid nitrogen, each sample holder is provided with a collar or other mechanical element to which a liquid nitrogen reservoir may be selectively coupled and uncoupled. While in the first storage dewar, each sample is pre-coupled to a liquid reservoir, so that when the sample is lifted out of the first storage dewar, the liquid reservoir is carried with the sample to keep the sample immersed in liquid nitrogen while it is in transition.
Once the sample is in position for x-ray crystallography to be performed, a controlled temperature gas stream, for example, nitrogen or helium, is directed over the sample and the liquid reservoir. The gas is refrigerated sufficiently to insure that the sample will remain at a controlled temperature while within the controlled temperature gas stream. A second robotic tool may then be used to grasp and decouple the liquid reservoir from the sample holder and to remove the liquid reservoir from the region of the sample so that x-ray crystallography can be performed on the sample. Prior to or during the x-ray crystallography process, the robotic arm provides all necessary spatial adjustments to the sample position, and orients or rotates the sample as necessary.
As an alternative to using a liquid reservoir to maintain the sample in an artificial environment while it is being moved from or to a storage dewar such as the first storage dewar, the controlled temperature gas stream may be provided through a jet which is attached to the first robotic arm or otherwise designed to travel with the first robotic arm. In this alternative configuration, the controlled temperature gas stream may be directed over the sample from the time it leaves the first storage dewar until the time it is replaced in the first storage dewar or otherwise released from the first robotic arm. Using such a configuration, the fluid reservoir would not be required, nor would the second robotic tool be required to couple and uncouple the fluid reservoir from the sample holder.
Those of skill in the art will recognize that performing x-ray crystallography on the sample requires an x-ray source and a detector, and that many variations and combinations of these devices are possible and known in the art. For example, the x-ray source can be an x-ray tube, a rotating anode, or a synchrotron source and will include beam conditioning optics including collimation or slits. A suitable detector will be any device capable of measuring diffraction events, including imaging plate detectors, CCD detectors, multiwire detectors, and digital pixel array detectors.
After testing, the second robotic tool is used to replace the liquid reservoir and to recouple it to the sample holder, so that the sample may be removed from the controlled temperature gas stream and remain at a controlled temperature. The robotic arm can then place the sample in a receiver dewar in a manner that allows for its continued identification, or can return the sample to the first storage dewar and replace it therein. Whether the sample is returned to the original dewar or placed in a second dewar is not critical to the functioning of this invention, and will be recognized by those of skill in the art as variable arrangements made for the convenience of the system""s users.